The invention relates to a process for the production of a DNA (desoxyribonucleic acid) comprising the genome characteristic of that of the B hepatitis virus. It also relates to DNAs of which a fragment is constituted by a double strand DNA corresponding to that of viral B hepatitis. In addition, it relates to vectors and compositions including such DNAs, for taking advantage of their biological properties. The invention relates to a nucleic acid comprising a nucleotide sequence capable of coding an immunogenic peptide sequence corresponding to the surface antigen of the virus of viral hepatitis B, and to the polypeptides and peptides obtained. It relates also to a process enabling such a nucleic acid to be obtained.
B hepatitis is a frequent viral disease, more particularly in tropical Africa, in Southeast Asia, and in the Far East where about 10% of the people are carriers of the surface viral antigen also designated as HBs antigen.
Though the infection is often manifested by an acute form without sequelae, it can also be at the origin of a chronic hepatitis, of cirrhosis, and even of fatal hepatic necrosis. This explains the importance of studies devoted to the biology of the virus, and the recent development of a vaccine whose efficiency has been demonstrated on patients and members of the personnel of hemodialysis centers (Ph. MAUPAS, A. GOUDEAU, P. COURSAGET, J. DRUCKER and Ph. BAGROS, Intervirol., 10, 1978, p. 196-208). The Dane particle (D. S. DANE, C. H. CAMERON and M. BRIGGS, Lancet. i. 1970, p. 695-698) is at present considered as the etiological viral agent. This particle, which can be detected by observation with the electron microscope, has a diameter of 42 nm. The patient""s serum in the preicteric phase contains up to 109 or even 1010 of it per milliliter. It possesses an envelope (Australia antigen or HBs antigen), a capsid (HBc antigen), an endogenic polymerase, and a DNA molecule (J. L. Melnick, G. R. DREESMAN and F. B. HOLLINGER, Sc. Amer., 237, 1977, p. 44-52). Under observation with the electron microscope, the genome appears as a bicatenary DNA ring possessing a monocatenary region, whose length varies from one molecule to the next (J. SUMMERS, A. O""CONNELL and I. MILLIMAN, Proc. Nat. Acad. Sc., 72, 1975, p. 4597-4601), (W. S. ROBINSON, Ann. Rev. Microbiol., 31, 1977). This ring is constituted by two intertwined linear molecules of unequal lengths (as shown diagrammatically in FIG. 1). It is the smallest viral genome known in mammals. The longest strand contains about 3,200 bases. The endogen polymerase DNA can be used to repair in vitro the single strand region (b1 in FIG. 1) of the shortest strand (T. A. LANDERS, H. B. GREENBERG and W. S. ROBINSON, J. Virol., 23, 1977, p. 368-376). All these very special properties of the Dane particle further increase the interest of studying the biology of this virus.
Electrophoretic analysis of the proteins of the envelope has shown the presence of 2 to 7 polypeptides of which the principal are called: polypeptide I and polypeptide II (PETERSON D. L., ROBERS I. M. and VYAS G. N. (1977) Proc. Nat. Acad. Sci., USA, 74 1530-1534, and PETERSON D. L., CHIEN D. Y., VYAS G. N., NITECHKI D., and BOND H. (1978). In Viral Hepatitis, ed. G. VYAS, S. COHEN and R. SCHMID, The Franklin Institute Press, Philadelphia, 569-573).
The Polypeptide I has a weight of 22,000 to 26,000 daltons. Polypeptide II is glycosylated and has a molecular weight of 28,000 to 30,000 daltons. The amino acid composition of these two polypeptides is very similar, the sequences which form, respectively, their 15 first amino acids (from the N-terminal end) and their last 3 amino acids are identical, so that the hypothesis has formulated that polypeptide II could differ from polypeptide I only by a glycosylation. Until now, the sequence of the I and II polypeptides themselves, and the location in the viral DNA of the sequence coding these peptides have not been done.
Study of the virus is, however, at present made particularly difficult by reason of the difficulties of supplies of serum containing Dane particles. Even a rich serum does not permit the preparation of large amounts of DNA (of the order of 1 xcex3 of DNA per volume of 500 ml of serum). It is hence necessary to collect serums of various origins corresponding to several genetic variants (J. P. SOULIER and A. M. COUROUCE-PAUTY, Vox Sang. 25, 1973, p. 212-235), which renders precarious a study of the primary structure of the genome. The presence of the single strand region makes difficult, moreover, the establishment of a physical map by restriction enzymes.
The problem of the isolation of relatively large amounts of viral particles attains a still increased importance, when it is desired to have available sufficient amounts of viral particles, more particularly of their HBs antigens, which appear to carry a surface antigen (protein) having vaccinating properties. The present methods of vaccination, if they have demonstrated their efficiency, are not however absolutely devoid of drawbacks. In particular, preparations of HBs, used as a vaccine, may contain antigen components coming from hepatic cells, which can be the origin of an autoimmune response (B. S. BLUMBERG, Science, 197, 1977, p. 17-24).
Study of the virus is also extremely difficult to the extent that no cell culture system is available enabling the propagation of the virus. This difficulty has already in part been overcome, more particularly as regards the ayw serotype. The whole DNA (genome) of the virus has been identified and cloned, notably in E. coli, after its previous insertion in the single EcoRI site of a xcex gt. WES. xcexB vector, according to the technique by FRITSCH A., POURCEL C., CHARNAY P., and TIOLLAIS P. (1978) C. R. Acad. de Paris, 287, 1,453-456).
Until now, the sequence of the I and II polypeptides themselves, and the location in the viral DNA of the sequence coding these peptides have not been done.
It is, therefore, an object of the invention notably to overcome these difficulties, more particularly to provide a process enabling the production of DNA of B hepatitis virus (or of the Dane particle), in sufficient amounts for the realization of the above-mentioned studies, and in a state of purity such that its use can be contemplated, even for therapeutic uses.
It is also an object of the invention to provide a much smaller DNA sequence than the viral DNA itself, containing the sequence adapted to code the peptide sequence endowed with immunogenic properties enabling, when it is introduced into the organism of a living host, to induce the formation by the latter of antibodies capable of protecting this same host subsequently with respect to the virus of viral hepatitis B, notably when the latter is in virulent state.
The invention takes advantage of the fact that the DNA of the Dane particle possesses, after in vitro xe2x80x9crepairxe2x80x9d in the presence of precursor nucleotides and of a polymerase, a single recognition site with regard to certain endonucleases, notably restriction enzymes, such as the enzyme EcoRI or Xho.
The process of the invention for producing a DNA comprising the genome characteristic of that of the DNA of the B hepatitis virus is characterized by the cloning in a bacterium of a double strand DNA, formed from the B hepatitis virus DNA, notably after repair of the latter in vitro as indicated above. This double strand DNA will be denoted below as DNA-HVB. Preferably, the polymerase used is endogenous polymerase of the B hepatitis virus itself.
Preferably, the DNA to be cloned has, previously, been cleaved by an endonuclease, such as defined above, notably by the restriction enzyme EcoRI.
The invention stems not only from the complete nucleotide analysis of the genome of the Dane particle,which the inventors have achieved, but to the idea that they have had for identifying the coding gene (called below xe2x80x9cS genexe2x80x9d) of the abovesaid polypeptides, to search in the complete nucleotide structure thus preestablished of the genome of the Dane particle, for those of the sequences of the nucleotides capable of coding the known proximal and terminal peptide sequences of these polypeptides.
To carry out the cloning, recourse is advantageously had to a vector, notably a phage or plasmid, in which the double strand DNA, previously cleaved at its single site, will have first been inserted.
By way of example of a phage enabling the easy cloning of the double strand DNA, first opened by EcoRI, may be mentioned xcexgtWES. xcexB (P. LEDER, D. TIEMEIER, and L. ENQUIST, SCIENCE, 196 (1977) pp. 175-177), which only comprises two EcoRI sites (EcoRI xcex1 and EcoRI xcex2). The latter enable the insertion of the whole of the DNA of the B hepatitis virus in the genome of this phage, instead and in place of the fragment inside this virus and previously situated between these two EcoRI sites.
It is naturally self-evident that any other vector comprising two EcoRI sites, or even a single EcoRI site, in a part unessential for its own replication, may be used for the same purposes.
Thus, the cloning process according to the invention can include the following essential steps:
the repair of the DNA of the B viral hepatitis, in the presence of precursor nucleotides and of a polymerase to form DNA-HVB;
the cleavage of the DNA-HVB by the enzyme selected, notably EcoRI;
the cleavage of the DNA of the vector, recovery of the portions of this DNA (two or three according as the vector includes one or two EcoRI sites), separation and isolation, notably by ultra centrifugation of the two parts of this DNA, which contained in particular, respectively, the head and tail genes of the phage and the replication genes of the phage (the two operations which precede being feasible simultaneously);
the mixing of these two parts of the DNA vector and of the DNA-HVB and their reaction in the presence of DNA-ligase, notably such as T4 DNA-ligase;
the transfection or transformation of a culture of host bacteria by the products obtained; and, after incubation of the culture,
the recovery of phages, the extraction of their DNA recombinants, denoted below by xcex-HVB1, which then contain DNA-HVB inserted in their genomes and, optionally, the treatment of the latter by the EcoRI enzyme and the isolation notably by ultracentrifugation, of the DNA-HVB, which is detectable by electrophoresis on an agarose gel, due to the fact that it migrates as the slowest fraction and the size of which can then be evaluated at about 3,200 pairs of bases.
Hence, novel products are obtained which are of direct use in several fields. The DNAs thus produced, before or after separation of the DNA-HVB, notably by cleavage with EcoRI, are usable as a probe for the in vitro diagnosis of the presence in biological samples of the B-hepatitis virus. To this end, these DNAs can be marked in any manner known in itself by a radio element. The use of such labeled DNAs is particularly advantageous in that it does not require considerable blood samples in persons in whom the presence of the B hepatitis virus is suspected.
By way of example, the interest which attaches to this use for study of contagious cases of B hepatitis or of the detection of the B hepatitis virus treated in hemodialysis centers, can be stressed. In the same way, this method of diagnosis is suited to the checking of blood samples which are to be involved in blood transfusion (or blood plasma or serum).
The invention also relates, by way of novel product, to the vectors in which a double strand DNA corresponding to that of viral hepatitis is inserted. In another mode of application of the invention, it is possible to induce the expression of the vectors as indicated above, in a bacterium, in order to induce the synthesis of a hybrid protein containing the HBs antigen in particular, for the study and for the preparation of vaccines with regard to B viral hepatitis.
With this in view, it may be advantageous to use as a vector a modified xcex bacteriophage comprising in combination:
mutations having the effect of preventing or retarding the expression of the late genes, particularly of regulating the production of the proteins necessary for encapsidation of this modified DNA in a bacterium notably E. coli, provided with suppressors of said mutations;
a DNA fragment comprising a part at least of the Z gene of E. coli and a promotor of the lactose operon (or of an analogous bacterial operon) inserted in a non-essential part of the genome of the phage; and
a site of cleavage by an endonuclease in the above-said part of the Z gene, to the exclusion of any other cleavage site by the same endonuclease in the above-said modified DNA.
Advantageously, the bacterial operon concerned is E. coli lactose operon, the mutations of the abovesaid late genes affect the Q and S genes and the single cleavage site is a EcoRI site.
Such a phage is disclosed in xe2x80x9cMolec. Gen. Gent.xe2x80x9d 170, pp. 171-178 (1979).
The manufacture of the above-said hybrid protein containing the protein corresponding to a DNA-HVB can then proceed as follows. It comprises infecting by said modified phage a bacterium, notably E. coli, not provided with suppressors of the mutations of the late genes of this phage, causing the bacterial strain, if necessary in the presence of a xcex2-galactosidase inducer, the hybrid protein being then recoverable from the cellular proteins formed.
If necessary, the vector used (whether it relates to the vector identified above by way of example or any other phage or plasmid vector) may be modified to ensure the reading in the correct phase of the DNA-HVB inserted in this vector. This can be carried out notably by resorting to the technique consisting of inserting in the vector concerned either two pairs, or four pairs of supplementary bases between the initiation point of the translation of the DNA fragment whose expression is sought and the first pair of bases of the recognition site proximal to the restriction enzyme, notably EcoRI, which is intended to constitute the linkage between the corresponding part of the vector and the proximal end of the DNA-HVB which must be inserted. The added pairs of bases must naturally be such that there are not introduced into the vector triplets of bases which would form a xe2x80x9cnonsensexe2x80x9d codon whose effect would be to interrupt the translation. There is thus obtained that the pairs of bases which in the first vector form respectively the first pairs of bases of each of the successively translated codons, become in at least a part of the two other vectors, respectively the second and third pairs (or vice versa) of the codons whose translation will be effected in the same host, previously transfected or transformed by these other vectors. It is thus possible to have a set of vectors enabling the three possible reading phases.
Starting from the first vector including an EcoRI site at a predetermined distance from the initiation point of the translation, it is possible to obtain one of the two other above-indicated vectors, for example by applying the process which comprises:
cutting the first vector by means of the EcoRI enzyme at the level of this site,
collecting that of the two phage fragments which comprises the initiation point of the translation,
trimming the monocatenary strand of its EcoRI cohesive end by means of a suitable endonuclease, for example S1 endonuclease,
recombining the thus modified fragment, at the level of the free end formed with a fragment such as that named xe2x80x9clinkerxe2x80x9d of the formula
xe2x80x83which itself possesses a recognition site for the EcoRI restriction enzyme,
producing the digestion of the modified vector fragment in the presence of EcoRI, which leads to the production of a vector fragment in which the first pair of bases of the EcoRI cohesive end is from then on shifted by two pairs of additional bases with respect to the position that it occupied previously with respect to the initiation point of the translation, and lastly
recombining this thus modified fragment with the other, notably in the presence of a DNA-ligase, notably T4 DNA-ligase.
The operation which has just been described can be repeated a second time to produce an additional similar shifting leading to the production of the third possible reading phase.
After insertion of the DNA-HVB in each of these three vectors, it is understood that one of these three vectors will be in any event adapted to be translated correctly by the suitable bacteria hosts, with consequently the production of a hybrid protein containing that which corresponds to DNA-HVB.
Other characteristics of the invention will appear also in the course of the description which follows of an example of cloning of the genome of the B hepatitis virus in the xcexgtWES.xcexB phage.